Method of microorganism detection using carbohydrate and lectin recognition

ABSTRACT

Methods of binding and detecting a microorganism on a solid substrate. The microorganism is bound on a solid substrate covalently bound to a capture agent having a saccharide moiety. A lectin capable of binding to the microorganism and the saccharide moiety of the capture agent is added to the sample to bind the microorganism on the solid substrate. Further provided are biosensor devices, such as a quartz crystal microbalance (QCM) device or a surface plasmon resonance (SPR) device, that incorporate the solid substrate for the detection of microorganisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit to U.S. Provisional Application Ser. No.60/850,561, filed Oct. 10, 2006, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This research was supported by grants from the National Institute ofHealth (NIH) (4R33 EB000672-02). The U.S. Government has certain rightsto this invention.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates generally to microorganism binding with asubstrate bound capture agent having a saccharide moiety and a lectin.In particular, the present invention relates to a method of binding amicroorganism on the substrate using the capture agent and lectin in anassay method and test kit.

(2) Description of the Related Art

Rapid methods for bacterial detection are essential in food, industrial,environmental monitoring, clinical diagnostics and biodefense to allowfaster decisions to be made with respect to food poisoning, watercontamination, the presence of disease and, therefore, treatmentoptions. Most conventional methods (e.g. plating and culturing,biochemical tests, microscopy, flow cytometry, luminescence) are timeconsuming, often requiring 1-2 days to obtain results. Although muchfaster detection methods such as immunosensors or DNA chips are becomingavailable, they have failed to gain wide acceptance due to the high userexpertise required, high cost of labeling reagents, and low stability ofantibody and DNA recognition elements. As a result, a rapid,quantitative, sensitive and specific method for one step bacterialdetection is highly sought after.

Cell surface carbohydrates (glycans) and adhesin molecules are majorcomponents of the outer surface of cells and are often characteristic ofthe cell types. Many adhesin molecules are lectins that havecarbohydrate binding activities. Glycans and adhesins are the firstinterface to the biotic and abiotic environment of the cell. Theinteractions of glycans with carbohydrate binding proteins (lectins) areperhaps the most significant and fundamental molecular recognitionevents in biological systems including bacterial pathogenesis, tumorcell metastasis, and inflammation. To understand the biological roles ofa particular carbohydrate or to evaluate a lectin adhesin as a diseasebiomarker, one must to determine when, where and how much a carbohydrateand/or lectin adhesin is expressed. The expression of carbohydratestructures changes dramatically during cell development and thecarbohydrates from different organisms display tremendous variations instructure and function. A similar situation exists for bacterial cellsurface lectin adhesin expression. As a result, the carbohydrate and/orlectin adhesin expression levels are extremely difficult to measure andpresent a formidable challenge for studying and characterizing theirroles in cell biology.

In recent years, advances in the fields of combinatorial carbohydratesynthesis and automated carbohydrate synthesis have made available agreat number of glycans for study. (³²Jelinek, R.; Kolusheva, S.“Carbohydrate Biosensors.” Chem. Rev. 2004, 104, 5987-6015) Carbohydratemicroarrays were developed to study the carbohydrate-cell interactionand to detect pathogens.⁽¹⁴⁾⁻⁽¹⁶⁾ Carbohydrate-arrays allow for theanalysis of protein carbohydrate interactions in a variety ofglycobiology systems, but they do not allow the direct examination ofchanges in glycosylation. Therefore, the bound lectin arrays that allowquick assess of bacterial cell surface carbohydrate compositions weredeveloped. Unfortunately similar to many protein arrays, they suffersome loss of binding activity in the coupling of lectin to the arrays.The large sizes of lectins also increase their susceptibility toproteases and encourage non-specific binding. Most previously reportedcarbohydrate and lectin arrays are one dimensional and use fluorescencelabel for detection. Fluorescence labeling of the bacteria cellsrequires additional steps. The presence of the labels themselves canintroduce additional interferences to the “true” binding process.Fluorescent detection can also suffer from high background fluorescencewhich may produce false positive results. U.S. Patent ApplicationPublication No. 2006/0014232 to Inagawa et al. teaches immobilization ofbiomolecules, provided with at least one tag, to a substrate. Thesubstrate has binding sites for the tags and activated reactive groupscapable of forming covalent bonds with the biomolecules. Thebiomolecules can be immobilized to prepare a sensor chip used forsurface plasmon resonance or quartz-crystal microbalance techniques. Theprior art does not teach the use of lectins to bind a microorganism tothe substrate.

While the related art teach bacterial detection methods, there stillexists a need for rapid, quantitative, sensitive and specificmicroorganisms analysis and detection method and test kit. Seereferences: ((¹⁷Nangia-Makker, P.; Conklin, J. Hogan, V.; Raz, A.“Carbohydrate-binding proteins in cancer, and their ligands astherapeutic agents.” Trends in Molecular Medicine 2002, 8, 187-192.)(¹⁸Stevenson, G.; Neal, B.; Liu, D.; Hobbs, M.; Packer, N. H.; Batley,M.; Redmond, J. W.; Lindquist, L.; Reeves, P. “Structure of theO-Antigen of Escherichia-Coli-K-12 and the Sequence of Its RfbGene-Cluster.” Journal of Bacteriology 1994, 176, 4144-4156); (¹⁹Lee, Y.C.; Lee, R. T. “Carbohydrate-Protein Interactions: Basis ofGlycobiology.” Accounts of Chemical Research 1995, 28, 321-7);(²¹Williams and Davies, Trends in biotechnology 2001, 19, 356-62);(²²Lindhorst, T. K. “Artificial multivalent sugar ligands to understandand manipulate carbohydrate-protein interactions.” Topics in CurrentChemistry 2002, 218, 201-235); (²³Houseman, B. T.; Mrksich, M. “Modelsystems for studying polyvalent carbohydrate binding interactions.”Topics in Current Chemistry 2002, 218, 1-44); (²⁴Liang, R.; Loebach, J.;Horan, N.; Ge, M.; Thompson, C.; Yan, L.; Kahne, D. “Polyvalent bindingto carbohydrates immobilized on an insoluble resin.” Proceedings of theNational Academy of Sciences of the United States of America 1997, 94,10554-10559); (²⁵Mathai Mammen, S.-K. C. G. M. W. “PolyvalentInteractions in Biological Systems: Implications for Design and Use ofMultivalent Ligands and Inhibitors.” Angewandte Chemie InternationalEdition 1998, 37, 2754-2794); (²⁶Shinohara, Y.; Hasegawa, Y.; Kaku, H.;Shibuya, N. “Elucidation of the mechanism enhancing the avidity oflectin with oligosaccharides on the solid phase surface.” Glycobiology1997, 7, 1201-1208); (²⁷Kolb, H. C.; Finn, M. G.; Sharpless, K. B.“Click Chemistry: Diverse chemical function from a few good reactions.”Angewandte Chemie-International Edition 2001, 40, 2004-+); (²⁸Ratner, D.M.; Adams, E. W.; Disney, M. D.; Seeberger, P. H. “Tools for glycomics:Mapping interactions of carbohydrates in biological systems.”Chembiochem 2004, 5, 1375-1383); (²⁹Mann, D. A.; Kanai, M.; Maly, D. J.;Kiessling, L. L. “Probing Low Affinity and Multivalent Interactions withSurface Plasmon Resonance: Ligands for Concanavalin A.” Journal of theAmerican Chemical Society 1998, 120, 10575-10582); (³⁰Ostuni, E.;Chapman, R. G.; Liang, M. N.; Meluleni, G.; Pier, G.; Ingber, D. E.;Whitesides, G. M. “Self-assembled monolayers that resist the adsorptionof proteins and the adhesion of bacterial and mammalian cells.” Langmuir2001, 17, 6336-6343); (³¹Fung, Y. S.; Wong, Y. Y. “Self-assembledmonolayers as the coating in a quartz piezoelectric crystal immunosensorto detect Salmonella in aqueous solution.” Analytical Chemistry 2001,73, 5302-5309); (³³Poxton, I. R. “Antibodies to lipopolysaccharide.”Journal of Immunological Methods 1995, 186, 1-15) and (³⁴Feizi, T.;Fazio, F.; Chai, W.; Wong Chi, H. “Carbohydrate microarrays—a new set oftechnologies at the frontiers of glycomics.” Current opinion instructural biology 2003, 13, 637-45).

OBJECTS

It is therefore an object of the present invention to provide a reliablemethod and test kit for analysis and detection of a microorganism. It isfurther an object of the present invention to provide an economicalassay to profile bacterial surface carbohydrate and lectin expression.These and other objects will become increasingly apparent by referenceto the following description and the drawings.

SUMMARY OF THE INVENTION

The present invention provides a method of attaching a microorganism toa solid substrate comprising: providing the substrate having a surfacecovalently bound to a capture agent having a saccharide moiety, themicroorganism in a sample, and an unbound lectin capable of binding tothe microorganism and to the saccharide moiety of the capture agent; andapplying the sample and a lectin to the substrate with the capture agenthaving the saccharide moiety, for a time to bind the lectin to thesaccharide moiety of the capture agent, and the lectin to themicroorganism to attach the microorganism to the solid substrate. Infurther embodiments, the saccharide moiety is a mannose basedsaccharide. In further still embodiments, the lectin is concanavalin A(ConA) and the microorganism is an E. coli species. In still furtherembodiments, the lectin is present in an amount that binds to multipleof lipopolysaccharides exposed on a cell wall of the microorganism andto the saccharide moieties. In further still embodiments, the lectin ispresent in an amount that binds to multiple of lipopolysaccharidesexposed on a cell wall of the microorganism and to the saccharidemoieties and wherein the saccharide moieties also bind to other parts ofthe microorganism.

The present invention further provides a method of detecting amicroorganism in a sample comprising: providing (1) a biosensor devicefor detecting the microorganism in the sample comprising a solidsubstrate having a surface covalently bound to a capture agent having asaccharide moiety, (2) the sample, and (3) an unbound lectin capable ofbinding to the microorganism and to the saccharide moiety of the captureagent; applying the sample and an unbound lectin to the solid substratewith the capture agent having the saccharide moiety, for a time to bindthe lectin to the saccharide moiety of the capture agent and the lectinto the microorganism to attach the microorganism to the solid substrate;and detecting the microorganism attached to the solid substrate with thebiosensor device. In further embodiments, the substrate is provided as acomponent in a quartz crystal microbalance (QCM) device, a surfaceplasmon resonance (SPR) device or an impedance device. In still furtherembodiments, a polyethylene glycol thiol is applied as a blocking agentto the solid substrate prior to applying the sample to reducenonspecific adsorption to the solid substrate. In further stillembodiments, the lectin is present in an amount which binds to multipleof lipopolysaccharides on an exposed cell wall of the microorganism.

The present invention further provides a kit for detection of amicroorganism in a sample comprising: a solid substrate with a captureagent having a saccharide moiety covalently bound to a surface of thesolid substrate; and an unbound lectin capable of binding themicroorganism and the capture agent, wherein when the microorganism inthe sample and the lectin capable of binding to the microorganism and tothe saccharide moiety of the capture agent are applied to the solidsubstrate, the lectin binds the microorganism and the saccharide moietyof the capture agent so as to bind the microorganism to the solidsubstrate for the detection. In further embodiments, the surface of thesolid substrate is metallic. In further still embodiments, the substrateis provided as a component in a quartz crystal microbalance (QCM)device, a surface plasmon resonance (SPR) device or an impedance device.In still further embodiments, the lectin is present in an amount whichbinds to multiple of lipopolysaccharides on an exposed cell wall of themicroorganism and to the saccharide moieties. In still furtherembodiments, the kit includes a blocking agent to prevent non-specificbinding. In further still embodiments, the substrate is a component of amultiple array of different lectins or polysaccharides bound to thesubstrate. In still further embodiments, the substrate is a component ofa multiple array of different lectins or polysaccharides bound to thesubstrate.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A, 1B and 1C are schematic representations of mannose SAMs and E.coli detection: 1A) Mannose SAM; 1B) Direct E. coli detection; 1C) Con Amediated E. coli detection.

FIG. 2 is a scheme illustrating the synthesis of linker[1-[(Methylcarbonyl)thio]undec-11-yl]-tetra(ethylene glycol).

FIG. 3 is a scheme illustrating the synthesis of (1-Mercaptoundec-11-yl)tetra (ethylene glycol) D-mannopyranoside conjugate.

FIG. 4 is a graph of the frequency change vs. time curve when FBS (5.2μg/ml), ECL (142 nM) and different concentrations of Con A solutionswere added sequentially to the mannose-QCM in 1.0 ml PBS buffer (pH=7.2)with 1 mM Mn²⁺ and 1 mM Ca²⁺.

FIG. 5 is a graph of the frequency change vs. time curve when themannose-QCM electrode was exposed to different concentrations of E. coliW1485 (2.9×10⁷, 9.8×10⁷, 1.6×10⁸, and 2.7×10⁸ cells/ml) in 1.0 mlstirred PBS buffer (pH=7.2) with 1 mM Ca²⁺ and 1 mM Mn²⁺.

FIG. 6 is a graph of the frequency change vs. time curve when themannose-QCM sensor was first exposed to 100 nM Con A, followed by theaddition of E. coli W1485 (7.5×10⁷ cells/ml) in 1.0 ml stirred PBSbuffer (pH=7.2) with 1 mM Ca²⁺ and 1 mM Mn²⁺.

FIG. 7A is the frequency change vs. time curve when mannose-QCMelectrodes were exposed to different concentrations of E. coli W1485from 7.5×10² to 7.5×10⁷ cells/ml in 1 ml stirred PBS with 1 mM Mn²⁺ and1 mM Ca²⁺ and 100 nM Con A. (The mannose-QCM was first exposed to 100 nMCon A solution for about two hours). FIG. 7B is a calibration curveshowing frequency shift vs. log of E. coli concentration.

FIG. 8 is a comparison of sensor specificity. Different electrodes wereexposed to 7.5×10⁷ cells/ml E. coli W1485: A) Con A pretreated mannoseelectrode with Con A in binding solution; B) Con A pretreated mannoseelectrode without Con A in binding solution; C) Con A-free mannosesurface and Con A-free binding solution; and D) Con A pretreated 210EscFv-cys electrode with Con A in binding solution. E): control antigen:1.4×10⁹ cells/ml Staphylococcus aureus were added to Con A pretreatedmannose electrode with Con A in binding solution. All the test chamberscontain 1.0 ml stirred PBS buffer (pH=7.2) with 1 mM Mn²⁺ and 1 mM Ca²⁺.

FIG. 9 is an SPR spectrum. A) mannose SAM, B) 3.7×10⁸ cells/ml E. coliW1485 was injected into mannose SAM for 60 min, C) 1.25 μM Con injectedinto mannose SAM for 40 min, D) 3.7×10⁸ cells/ml E. coli W1485 wasinjected to the Mannose/Con A surface for 60 min, E) the mixture of1.9×10⁸ cells/ml E. coli W1485 and 1.25 μM Con A injected to the Con Apretreated Mannose SAM for 70 min.

FIG. 9A is the light intensity change with time after the injection of1.9×10⁸ cells/ml E. coli W1485 and 1.25 μM Con A mixture onto the Con Apretreated Mannose SAM. The wavelength of the SPR biosensor light sourcewas 650 nm and the refractive index of its prism was 1.79. The samplechamber of the SPR biosensor was thoroughly washed before recording thespectrum, and filled with 200 μl PBS with 1 mM Ca²⁺, 1 mM Mn²⁺ for eachof the experiments.

FIG. 10 is the molecular formula of a synthesized lipid mannose-SH usedas a capture reagent for a surface plasmon resonance (SPR) QCM andElectrochemical Impedance Spectroscopy (EIS) biosensors.

FIG. 11 is a three-dimensional model of the synthesized mannose-SHmolecule used as the capture reagent for a surface plasmon resonance(SPR) biosensor. Black is oxygen and white is CH.

FIG. 12A is a graph illustrating mannose-SH binding to a gold surface ofa surface plasmon resonance biosensor while the liquid in the chamber isethanol. During measurements, the incident angle φ was fixed at 26.5°.FIG. 12B is a graph showed the SPR spectra shift caused by Mannose-SHlipid in ethanol.

FIG. 13A and FIG. 13B are graphs illustrating protein Concanavalin A(Con A) in PBS buffer binding to the mannose-SH immobilized on the goldsurface of the surface plasmon resonance biosensor. During measurementsfor FIG. 13A, the incident angle φ was fixed at 29.0°.

FIG. 14A and FIG. 14B are graphs illustrating E. coli w1485 binding tothe Con A surface (immobilized on the mannose-SH lipid monolayer on thegold surface of the surface plasmon resonance biosensor if there areConA molecules in the liquid. During measurements for FIG. 14A, theincident angle φ was fixed at 28.0°.

FIG. 15 is a schematic diagram of 1 and 2 dimensionalcarbohydrate-lectin microarrays.

FIG. 16 is a schematic representation of various Carbohydrate SAM andglycopolymer fabrications: A) Studying the interaction of alpha-Galcarbohydrate antigen and proteins by Quartz-Crystal Microbalance (QCM)JACS, 2003, 125, 9292; B) Studying of Carbohydrate-Protein Interactionsby “Clicked” Carbohydrate Self-Assembled Monolayers Anal. Chem. 2006;78; 2001; C) Enzymatic Synthesis of Oligosaccharides on CarbohydrateSelf-Assembled Monolayers, in preparation; D) Cross-linkedSurface-Grafted Glycopolymer for Multivalent Recognition of Lectin,Anal. Chem. in review; E) Alkanethiol Containing Glycopolymers: A Toolfor the Detection of Lectin Binding, Bioorganic & Medicinal ChemistryLetters, in revision.

FIG. 17 QCM real time ΔF vs. time curve; (A) Adding E. coli/Con Amixture to mannose sensor (pH. 7.2); (B-D) Adding E. coli to con Atreated mannose sensor at different pH; (E) Control: Adding E. coli tocon A-modified surface.

FIG. 18 Binding pattern of E. coli 1485 and 086 on mannose-sensor usingConA or ECL lectin mediator.

FIG. 19 scFv biointerface for S aureus detection. QCM real time ΔF vs.time curve.

FIG. 20 is a schematic diagram of a “Click” chemistry fabricationstrategy.

FIGS. 21A and 21B are carbohydrate-lectin 2D array for bacterialscreening (21A); illustrative 2-D carbohydrate and lectin expressionpatterns (21B).

FIG. 22 is a synthetic scheme showing oligosaccharides are synthesizedfrom azido lactose.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications, government publications, governmentregulations, and literature references cited in this specification arehereby incorporated herein by reference in their entirety. In case ofconflict, the present description, including definitions, will control.

High percentages of harmful microbes or their secreting toxins bind tospecific carbohydrate sequences on human cells at the recognition andattachment sites. A number of studies also show that lectins react withspecific structures of bacteria and fungi. We take advantage of the factthat a high percentage of micro-organisms have both carbohydrate andlectin binding pockets at their surface. It is demonstrated here for thefirst time that a carbohydrate non-label mass sensor in combination withlectin-bacteria recognition can be used for detection of high molecularweight bacterial targets with remarkably high sensitivity and enhancedspecificity. A functional mannose self assembled monolayer incombination with lectin Con A was used as molecular recognition elementsfor the detection of E. coli W1485 using Quartz Crystal Microbalance(QCM) as a transducer. The multivalent binding of Con A to the E. colisurface receptor (lipopolysaccharide (LPS)) favors the strong adhesionof E. coli to mannose modified QCM surface by forming bridges betweenthese two. As a result, the contact area between cell and QCM surfaceincreases and leads to rigid and strong attachment that enhances thebinding between E. coli and the mannose receptor. The results show asignificant improvement of the sensitivity and specificity ofcarbohydrate QCM biosensor with a detection limit of a few hundredbacterial cells and a linear range from 7.5×10² to 7.5×10⁷ cells/mL thatis four decades wider than the mannose alone QCM sensor. The change ofdamping resistances for E. coli adhesion experiments was no more than1.4%, suggesting that the bacterial attachment was rigid, rather than aviscoelastic behavior. Little non-specific binding was observed forStaphylococcus aureus and other proteins (Fetal Bovine serum, Erythrinacristagalli lectin). This approach not only overcomes the challenges ofapplying QCM technology for bacterial detection but also increases thebinding of bacteria to their carbohydrate receptor through bacterialsurface binding lectins that significantly enhanced specificity andsensitivity of QCM biosensors. Combining carbohydrate and lectinrecognition events with an appropriate QCM transducer yields sensordevices highly suitable for the fast, reversible and straightforwardon-line screening and detection of bacteria in food, water, clinical andbiodefense areas.

The term “saccharide” as used herein refers to a carbohydrate, includingmonosaccharides, disaccharides, and polysaccharides.

The term “microorganism” as used herein refers to any microorganism,including but not limited to, bacteria and fungi.

The term “bacteria” as used herein refers to include both gram-positiveand gram-negative bacteria.

The term “gram-negative bacteria” as used herein refer to anygram-negative bacteria such as, but not limited to, proteobacteria,including Escherichia coli, Salmonella, Vibrio, Helicobacter and otherEnterobacteriaceae, Pseudomonas, Moraxella, Helicobacter,Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella andothers. Gram-negative bacteria also include cyanobacteria, spirochaetes,green sulfur and green non-sulfur bacteria. Gram-negative cocci include,but are not limited to, organisms that cause sexually transmitteddisease (Neisseria gonorrhoeae), meningitis (Neisseria meningitidis),and respiratory symptoms (Moraxella catarrhalis). Gram-negative bacilliinclude, but are not limited to, baccilli causing respiratory problems(Hemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila,Pseudomonas aeruginosa), urinary problems (Escherichia coli, Proteusmirabilis, Enterobacter cloacae, Serratia marcescens), andgastrointestinal problems (Helicobacter pylori, Salmonella enteritidis,Salmonella typhi).

The term “lectin” as used herein refers to any molecules includingproteins, natural or genetically modified, that interact specificallywith saccharides (ie. carbohydrates). While the examples herein refer toa natural plant lectin, the term “lectin” herein refers to lectins fromany species, including but not limited to plants, animals, insects andmicroorganisms, having a desired carbohydrate binding specificity.Examples of plant lectins include, but are not limited to, theLeguminosae lectin family, such as ConA, soybean agglutinin, and lentillectin. Other examples of plant lectins are the Gramineae and Solanaceaefamilies of lectins. Examples of animal lectins include, but are notlimited to, any known lectin of the major groups S-type lectins, C-typelectins, P-type lectins, and I-type lectins.

The term “SPR” as used herein refers to a surface plasmon resonance. AnySPR device can be used in the present invention including, but notlimited to, a Biocore system (GE Healthcare) or the SPR biosensor deviceas described in U.S. patent application Ser. No. 11/581,260 to Xiao andZeng, filed Oct. 10, 2006.

The term “QCM” as used herein refers to a quartz crystal microbalance.Any quartz crystal microbalance devices can be used in the presentinvention including, but not limited to QCM devices available fromMaxtek Inc. of Santa Fe Springs, Calif. Other QCM devices which can beused in the present invention are described in U.S. Pat. No. 4,236,893to Rice, U.S. Pat. No. 4,242,096 to Oliveira et al., U.S. Pat. No.4,246,344 to Silver III, U.S. Pat. No. 4,314,821 to Rice, U.S. Pat. No.4,735,906 to Bastiaans, U.S. Pat. No. 5,314,830 to Anderson et al., U.S.Pat. No. 5,932,953 to Drees et al., and U.S. Pat. No. 6,087,187 toWiegland et al., U.S. Pat. No. 6,890,486 to Penelle, U.S. Pat. No.6,848,299 to Paul et al., U.S. Pat. No. 6,706,977 to Cain et al., U.S.Pat. No. 6,647,764 to Paul et al., U.S. Pat. No. 6,492,601 to Cain etal., U.S. Pat. No. 6,439,765 to Smith, U.S. Pat. No. 6,190,035 to Smith,U.S. Pat. No. 6,106,149 to Smith, U.S. Pat. No. 5,885,402 to Esquibel,U.S. Pat. No. 5,795,993 to Pfeifer et al., U.S. Pat. No. 5,706,840 toSchneider, U.S. Pat. No. 5,616,827 to Simmermon et al., U.S. Pat. No.5,484,626 to Storjohann et al., U.S. Pat. No. 5,282,925 to Jeng et al.,U.S. Pat. No. 5,233,261 to Wajid, U.S. Pat. No. 5,201,215 to Granstaffet al., U.S. Pat. No. 4,999,284 to Ward et al., and U.S. Pat. No.4,788,466 to Paul et al. Examples of control circuitry for quartzcrystal microbalances and methods for detecting materials usingpiezoelectric resonators are described in U.S. Pat. No. 5,117,192 toHurd and U.S. Pat. No. 5,932,953 to Drees et al. Each of the abovereferences are hereby incorporated herein by reference in theirentirety.

Carbohydrates cover cell surfaces and the interaction of carbohydrateswith their surrounding environment is one of the most fundamentalmolecular recognition events. The interaction of carbohydrates (alsocalled ligands or epitopes) with their corresponding proteins (alsocalled receptors) involves in cell/cell recognition, invasion ofvirus/bacteria/toxins, antibody recognition, hormonal action and manyother physiological and pathological processes. Therefore, carbohydrateligands or epitopes in principle can be used as sensing elements todetect and monitor a variety of recognition events.

A high percentage of harmful bacteria and their secreted toxins bindspecific carbohydrate sequences on the surface of human cells at theinitial recognition and attachment site. Recently it has become clearthat type 1 fimbriae present on the surface of Enterobacteriaceae areresponsible for mannose- and mannoside-binding activity. Escherichiacoli (E. coli), especially E. coli 0157:H7 is a significant cause offood contamination and food-borne illness. Preventing food contaminationfrom E. coli requires effective risk management controls at all stagesof the food production continuum. Proper hygiene and controls must beincorporated into all processes, from agricultural production to finalpreparation and serving. In order to fulfill this requirement thedevelopment of systems for quick, one-step detection of E. coli andother harmful microorganisms is crucial.

Knowing the nature of bacteria and their host cell invasion processes,where carbohydrate interactions play an important role in the stabilityand rigidity of saccharide assemblies, it seems logical to usecarbohydrate structures as receptor elements in pathogen detectionschemes.

However, carbohydrate-protein interactions are often weaker thanprotein-protein interactions, by perhaps a factor of 10²-10³ based ontypical antibody equilibrium dissociation constants (K_(D)).Additionally, it is commonly accepted that apart from toxins andbacteria, many other endogenous and exogenous proteins could recognizethe carbohydrate ligand, which could lead to high cross activity. Theimportant question is whether these prevalent interactions could providea suitable alternative to the use of antibodies or nucleic acid fordetection and identification.

There are few examples to-date of carbohydrates actually being employedin biological detection systems. Ganglioside-bearing liposomes have beenused for the identification and differentiation of cholera toxin,botulinum toxin C fragment, and tetanus toxin C fragment, for whichsensitivity was as low as 1 nM. Detection of cholera toxin by G_(M1)ganglioside recognition has also been investigated using flow cytometry,and fluorescence self-quenching as a signal-transduction mechanism, withsensitivities of 10 and 50 pM, respectively. A biosensor based ondetection of the disaccharide Gal(α1-4)Gal, has been used foridentification of uropathogenic p-fimbriated E. coli, while influenzavirus has been detected calorimetrically with glycopolythiophenescontaining sialic acid. Overall, the examples above show that thecarbohydrate ligands or epitopes in principle have the potential to actas sensing elements to detect a variety of recognition events which canlead to early detection for cell invasion, tissue destruction and systeminfection. However, up to now, the optimal conditions for theirapplication as receptor elements, particularly when a non-labeledtransducer is selected as the transduction mechanism, have yet to bedeveloped.

A non-label biosensor such as Quartz Crystal Microbalance (QCM) orSurface Plasmon Resonance (SPR) offers significant advantages overcurrent labeled techniques. Being label free, it dispenses with the timeand cost demanding labeling step, and also eliminates any possibleinterference of the “true” binding process due to the presence of thelabels. As such, our method allows the analysis of real timeinteractions of any biomolecule and is able to deliver high quality,high information content analysis for complicated biological recognitionprocesses. Commercial developments of such techniques have been slowmostly due to a failure to appreciate the scale of the analyticalproblems and the inconsistent results sometimes obtained.

QCM is a mass sensor and is ideal for detecting analytes of highmolecular weights. It gives a response that characterizes the bindingevent between the analyte to be detected and a sensing layer, which isimmobilized on the surface of the QCM transducer. The resonant QCMfrequency depends on the mass attached to the quartz crystal surfaceaccording to the Sauerbrey relationship, Δf=−2Δmnf₀²/[A(μ_(q)ρ_(q))^(1/2)], where n is the overtone number, μ_(q) is theshear modulus of the quartz (2.947×10¹¹ g/(cm·sec²), and ρ_(q) is thedensity of the quartz (2.648 g/cm³), which assumes the foreign mass isstrongly coupled to the resonator. Methods based on the use ofpiezoelectric crystal devices have been developed for immunoassays,bacterial detection¹⁻¹⁰ and virus and toxin detection. Due to thenon-rigid nature of bacterial cells, researchers are still skepticalabout the potential of piezoelectric mass sensing devices for detectionof bacteria. QCM measures only those materials that are acousticallycoupled to the sensor surface and requires the surface layer to berigid.¹¹ Bacterial binding often involves energy dissipation due tointernal friction or trapping of water by the cells, which cause dampingof the oscillation of the crystals. As a result, surface chemistry needsto be developed to ensure that the bacteria are strongly attached on theQCM transducer surface, which is not a trivial task. For example, inmany bacteria, the carbohydrate binding lectins are usually in the formof fimbriae (or pili). The pili typically have a diameter of 3-7 nm andcan extend 100-200 nm in length. The bulk of the fimbrial filament ismade up of polymers of the major subunit, which thus plays a structuralrole. Only one of the subunits, usually a minor component of thefimbriae, possesses a carbohydrate combining site and is responsible forthe binding activity and sugar specificity of the fimbriae. For example,in type 1 fimbriae, which are made up of hundreds of subunits of fourdifferent kinds, the subunit (MW 29-31 KDa) is present in small numbersat intervals along the fimbrial filament and at the distal tip. However,only these subunits appear to be able to mediate mannose-sensitiveadhesive interactions, whereas the subunits at the other positions areinaccessible to the ligand. Consequently, binding is generally of lowaffinity and not rigid. But since the adhesions and the receptors oftencluster in the plane of the membrane, the resulting strength of theinteraction can be quite strong.

Bacteria cells have rigid cell wall so they are more rigid thaneukaryotic cells. Additionally, the cell wall composes ofpolysaccharides and peptides, for example lipopolysaccharides (LPS),which are often unique to specific bacterial strains (i.e. sub-species)and are responsible for many of the antigenic properties of thesestrains. LPS consist of a hydrophobic domain known as Lipid A anchoredin the membrane, a “core” oligosaccharide, and a polysaccharide(O-antigen). The O-antigens, being at the utmost cell surface, are atthe interface between the bacterium and its environment, and areimportant virulence factors and antigens for many pathogenic bacteria.Many bacteria are sub-classified by the O-antigen on their surface. Forexample, E. coli has 166 different O-antigens reported so far. Thesurface polysaccharide structures of several other bacteria have alsobeen structurally defined. Therefore, LPS of gram-negative bacteria isthe most striking character for bacteria providing the selectivespecificity needed for the lectin recognition.

A completely new approach has been developed that uses both theselective lectin-O-antigen recognition and carbohydrate-proteinrecognition for bacterial detection that provides enhanced specificityand needed rigidity for non-label QCM biosensors. Specifically, amannose receptor immobilized on the gold QCM sensor surface is used andit is used to detect E. coli W1485 as a model system. As discussedearlier, type-1 fimbriae present on the surface of most E. coli strainsare responsible for mannose- and mannoside-binding activity. Accordingto the studies conducted by Otto and coworkers,¹⁰ the direct adhesion ofthe fimbriated E. coli onto the mannose immobilized QCM surface, isquite flexible, and there might be water layers trapped between thebacteria and QCM surface. This non-rigid binding may cause damping ofthe oscillation. Given the fact that E. coli and other bacteriaespecially the gram-negative bacteria, have chemically distinct surfacelipopolysaccharides (LPS) that could be recognized by specific lectins,Concanavalin A (Con A) is preferably first bound to the E. coli W1485.ConA, isolated from Jack bean (Canavalia ensiformis) is the most widelyused and well-characterized mannose binding lectin. Con A can aggregateon specific terminal carbohydrates of bacterial surface LPS withdifferent binding ability. The multivalent binding of Con A to the E.coli surface receptor favors the strong adhesion of E. coli to mannoseimmobilized on the QCM surface. As a result, Con A increases the contactarea between the E. coli cell and the mannose ligands immobilized on thegold QCM surface. This leads to a relatively rigid and strong attachmentthat enhances and amplifies the binding between E. coli and the mannosereceptor (see FIG. 1). Our approach not only overcomes the challenges ofapplying QCM technology for bacterial detection but also increases thebinding of bacteria to their carbohydrate receptor through bacterialsurface binding lectins, thus significantly enhanced specificity andsensitivity of QCM biosensors.

Additionally, the advantage of SAM and the synthetic strength ofmolecular design here combined by building a functional mannose coatingto prevent non-specific adsorption. The linker for the functionalmannose coating consists of two parts: the polyethylene glycol([OCH₂CH₂]_(n)OH, n=4) portion and the saturated alkyl portion(R═(CH₂)₁₁) (FIG. 1). The polyethylene glycol part is linked withmannose, while the alkyl portion is terminated with —SH group, whichwill anchor the molecule on the Au surface of the QCM or SPR sensor.mPEG-thiol was used as a blocking reagent to reduce the nonspecificadsorption. Previous research shows that monolayers terminated in shortoligomers of the ethylene glycol group ([OCH₂CH₂]_(n)OH, n=3-6) preventthe adsorption of proteins under a wide range of conditions.¹² Thisalternative approach has several advantages over current methodsincluding well-defined surface chemistry, relative stability and facileformation into defined supramicron to nanometer-scale two-dimensionalpatterns.

Materials and Methods.

Chemicals and Materials: 1H and ¹³C NMR spectra were recorded on aVXR400 NMR and a Varian Unity 500 MHz spectrometers. Mass spectra wererun on Kratos MS-80 and Kratos MS-50 instruments. Thin-layerchromatography was conducted on precoated Whatman K6F silica gel 60 ÅTLC plates with a fluorescent indicator. EM Science silica gel 60Geduran (230-400 Mesh) was used for column chromatography. ConcanavalinA (Con A), and lectin from Erythrina cristagelli (ECL) were purchasedfrom Sigma (Sigma-Aldrich, St. Louis, Mo.). Escherichia coli, alambda-derivative of E. coli strain W1485 (ATCC® 12435™) was obtainedfrom ATCC. mPEG-thiol was purchased from NEKTAR. Phosphate bufferedsaline and fetal bovine serum were obtained from Gibco.

Bacterial strain and culture: The pure culture of Escherichia coli, alambda-derivative of E. coli strain W1485 (ATCC® 12435™) was grown inATCC®294 broth (tryptone with NaCl) at 37° C. for 24 hr in a shakingincubator. The viable cell number was determined by conventional agarplate counting.

Phosphorus tribromide (5.3 g, 19.6 mmol, 1.84 ml) was added dropwise toa solution of 10-Undecen-1-ol (10 g, 58.7 mmol) in diethyl ether (100ml) at −78° C. over a period of 10 min and then the mixture was allowedto warm to room temperature and stirred under Ar overnight. The reactionmixture was quenched with water and the layers were separated. Theaqueous layer was extracted with ether (5×30 ml) and the combinedorganic layers were washed with brine and dried over sodium sulfate.After evaporation of the solvent in vacuo, the crude product waspurified by chromatography column on silica gel (hexane) to give pureproduct 8.2 g (yield: 60%). ¹H NMR (CDCl₃) δ 5.85-5.75 (m, 1H),5.01-4.91 (m, 2H), 3.37-3.41 (t, 2H, J=7.2 Hz), 2.06-1.99 (m, 2H),1.88-1.81 (m, 2H), 1.39 (br, 2H), 1.29 (br, 10H). ¹³C NMR (CDCl₃) δ139.4, 114.3.3, 34.2, 34.1, 33.0, 31.8, 29.6, 29.3, 29.1, 29.0, 28.4.

A mixture of 1 ml of 50% aqueous sodium hydroxide (0.012 mol) and 23.4 gof tetra (ethylene glycol) (0.12 mol) was stirred for 0.5 h in an oilbath at 100° C. under Ar, and 2.8 g of 11-bromoundec-1-ene (0.012 mol)was then added. At the completion of the reaction as indicated by theTLC analysis, the reaction mixture was cooled and extracted severaltimes with hexane. The combined organic layers were washed with brineand dried over sodium sulfate. After evaporation of the solvent invacuo, the crude product was purified by chromatography column on silicagel (hexane/ethyl acetate, 1:1) to give pure product 2.5 g (yield: 60%).¹H NMR (CDCl₃) δ 5.82-5.72 (m, 1H), 4.97-4.87 (m, 2H), 3.68-3.64 (t, 2H,J=4.8 Hz), 3.62-3.54 (m, 14H), 3.42-3.39 (t, 2H, J=7.2 Hz), 2.01-1.97(m, 3H), 1.55-1.50 (m, 2H), 1.35-1.24 (m, 12H). ¹³C NMR (CDCl₃) δ 139.5,114.4, 72.8, 71.8, 70.8, 70.7, 70.5, 70.2, 34.0, 29.8, 29.7, 29.6, 29.3,29.1, 26.3. MS ES⁺ m/z 369.28 (M+Na).

Solutions of Undec-1-en-11-yltetra (ethylene glycol) (2.66 g, 7.7 mmol)in MeOH (40 ml) containing 3 equivalent of thioacetic acid (1.6 ml) and5 mg AIBN were purged with Ar for one hour (1 h), then the mixture wasirradiated under standard conditions (medium pressure mercury lamp,Pyrex glass filter) until the disappearance of the starting materials asindicated by TLC analysis. At the completion of the reaction, thesolvent was removed in vacuo, the crude product was purified bychromatography on silica gel (hexane/ethyl acetate 2:1) to give pureproduct 2.3 g (yield: 70%). ¹H NMR (CDCl₃) δ 3.62-3.46 (m, 16H),3.36-3.33 (t, 2H, J=6.4 Hz), 2.77-2.73 (t, 2H, J=7.2 Hz), 2.21 (s, 3H),2.19 (m, 2H), 1.49-1.44 (m, 2H), 1.16 (br, 14H). ¹³C NMR (CDCl₃) δ196.1, 72.8, 71.7, 70.7, 70.4. 70.1, 61.7, 30.7, 29.7, 29.6, 29.5, 29.2,28.9, 26.2. MS ES⁺ m/z 445.34 (M+Na).

Linker [1-[(Methylcarbonyl)thio]undec-11-yl]-tetra(ethylene glycol)(0.889 g, 2.1 mmol) in 20 ml anhydrous dichloromethane, HgBr₂ (0.94 g,2.4 mmol) and Hg(CN)₂ (0.65 g, 2.4 mmol) were added to a previouslyflame-dried flask containing 1 g 4 Å molecular sieve. After the mixturewas stirred for one hour (1 h) under Ar,2,3,4,6-tetra-O-acetyl-α-D-mannosyl bromide (1.25 g, 2.94 mmol) wasadded to the mixture. The mixture was stirred in the dark at ambienttemperature until the completion of the reaction as indicated by TLCanalysis. The resulting mixture was passed through a Celite packed glassfunnel, and washed with saturated NaHCO₃ and brine. The organic phasewas dried over anhydrous Na₂SO₄ and concentrated in vacuo. The resultingresidue was purified by chromatography column on silica gel(hexane/ethyl acetate, 1:1) to give the product 0.96 g (yield: 61%). ¹HNMR (CDCl₃) δ 5.30-4.80 (m, 3H), 4.68 (br, 1H), 4.11-4.05 (m, 1H),3.93-3.88 (m, 2H), 3.68-3.37 (m, 16H), 3.24 (t, 2H, J=7.2 Hz), 2.67-2.64(t, 2H, J=6.8 Hz), 2.12 (s, 3H), 1.95 (s, 3H), 1.90 (s, 3H), 1.85 (s,3H), 1.84 (br, 2H), 1.78 (s, 3H), 1.36 (br, 2H), 1.07 (br, 14H). ¹³C NMR(CDCl₃) δ 195.7, 170.5, 169.9, 97.7, 71.4, 70.5, 70.0, 69.5, 69.1, 68.4,67.3, 66.1, 62.7, 62.4, 60.3, 29.6, 29.5, 29.4, 28.7, 20.8. MS ES⁺ m/z775.47 (M+Na).

Anhydrous methanol (MeOH) was added to[1-(Methylcarbonyl)thio]undec-11-tetra(ethylene glycol)2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (0.814 g, 1.08 mmol). Afterthe solution was flushed with argon (Ar) for 20 min., NaOMe (0.583 g,10.8 mmol) was added, the reaction mixture was stirred under Ar at roomtemperature (r.t.) until the completion of the reaction as indicated bythe TLC analysis. Dowex cation exchange resin (H form) was added toadjust the pH to 6-7, the resin was filtered off and the filtrate wasconcentrated in vacuo. The resulting residue was purified bychromatography (CH₂Cl₂/MeOH, 5:1) to afford the product (8) 526 mg(yield: 90%). ¹H NMR (CD₃OD) δ 4.79 (d, J=1.8 Hz, 1H), 3.84 (dd, J=4.0Hz, 1H), 3.80-3.81 (m, 2H), 3.72 (m, 1H), 3.71 (d, J=13.2, 2H), δ3.65-3.64 (m, 16H), 3.36-3.33 (t, 2H, J=6.4 Hz), 2.77-2.73 (t, 2H, J=7.2Hz), 2.21 (s, 3H), 2.19 (m, 2H), 1.49-1.44 (m, 2H), 1.16 (br, 14H) ¹³CNMR (CD₃OD) δ 100.5, 73.4, 70.4, 70.2, 69.9, 67.4, 66.6, 61.7, 34.1,29.5, 29.1, 28.3, 26.0, 23.8. ESI m/z 541.48.

Quartz Crystal Microbalance: A non-polished gold quartz crystal(International Crystal Manufacturing Co. Inc.) was mounted in a(custom-made Kel-F cell [please detail]). It was cleaned three timesusing a mixture of concentrated nitric acid and sulfuric acid (1:1 v/v),biograde water and ethanol in series, and then the cell was dried usinga nitrogen stream. The frequency of the electrode was measured in PBS(pH 7.2). One side of the gold quartz crystal was incubated in asolution of 4 mg/ml mannose thiol linker conjugate in anhydrous ethanolat 4° C. overnight. After incubation, the gold surface was washed withethanol and biograde water and then dried under nitrogen to give mannoseSAMs. Any remaining sites on the mannose modified QCM surface wereblocked using 3 mg/ml mPEG-thiol (PI-03D-18, NEKTAR) for 6 hr. Thechanges in frequency and damping resistance of the QCM were monitoredsimultaneously using a network/spectrum/impedance analyzer (Agilent4395A) controlled by a PC via an Intel card.

Surface Plasmon Resonance: A surface plasmon resonance (SPR) biosensorwas used to detect bacterial attachment. The light source was a06-DAL-103 model semiconductor diode laser (Melles Griot Inc.,California) with 4 mW power output, as described in U.S. patentapplication Ser. No. 11/581,260 to Xiao and Zeng, filed Oct. 10, 2006,incorporated herein by reference in its entirety. The wavelength of thepolarized laser was 650 nm. The refractive index of the glass prism(ZF7) used is 1.79. A series-600 angular sensor (Trans-Tek Inc.,Ellington, Conn.) is used to measure laser incident angles. With afifteen volt direct current (15V DC) input, the capacitance angle sensorcan give an angle signal output of 100 mV/Degree. Also a solar cell isused to convert reflected light intensity into electric voltage. The twoanalog signals from the angular sensor and the solar cell are convertedinto 16-bit digital signals by an USB-1608FS data acquisition module(Measurement Computing Corporation, MA), which is connected to a PCthrough a USB cable. The hardware of the SPR biosensor is controlled bya program written in Labview® 8.0.

Results and Discussion

Synthesis: Synthesis of linker[1-[(Methylcarbonyl)thio]undec-11-yl]-tetra(ethylene glycol) is shown inFIG. 2. Treatment of the commercially available ω-undecylenyl alcoholwith PBr₃ and then reaction with tetraethylene glycol givesundec-1-en-11-yl tetra-(ethyleneglycol) (3), which is then treated withthiolacetic acid under photolysis condition initiated by AIBN to providethe desired linker [1-[(methylcarbonyl) thiol) undec-11-tetra(ethyleneglycol) (4). The synthesis of (1-Mercaptoundec-11-yl) tetra (ethyleneglycol) D-mannopyranoside conjugate is shown in FIG. 3. Connection ofthe linker (4) to mannose via a glycosylation reaction promoted by HgBr₂and Hg(CN)₂ and then deprotection provides the target compound (8).

Detection of Con A by mannose-QCM: Con A has identical subunits of 237amino acid residues (M.W.: 26,000). At neutral pH, Con A ispredominantly tetrameric with optimal activity. At pH 4.5-5.6, Con Aexists as a single dimer. Two metal ions (Mn²⁺ and Ca²⁺) can bind to ConA; both must be present for carbohydrate binding. Therefore, Con A hasbeen used to examine the mannose-QCM sensor performance.

To examine our mannose sensor's specificity, erythrina cristagallilectin (ECL), a galactose-specific legume lectin, was used as a negativecontrol. Fetal bovine serum (FBS), the most widely used serum in theculturing of cells, tissues and organs, was also selected as a negativecontrol. As shown in FIG. 4 Insertion, there were negligible frequencychanges for the addition of FBS and ECL. This result shows that themannose-QCM sensor is antigen specific. After exposure to FBS and ECL,Con A at different concentrations was consecutively added to the sensor(FIG. 4). This study demonstrates that the mannose-QCM sensor has highsensitivity and specificity for binding with the Con A even afterexposure of the sensor surface in a complex matrix (i.e. FBS and ECL).

The apparent binding affinity for Con A binding to the mannose QCMsurface was estimated by using the Langmuir adsorption model. Accordingto the following equation (Equation 1), the mass change at equilibriumwas related to the original concentration of Con A.

$\begin{matrix}{{{\lbrack{ConA}\rbrack + \left\lbrack {{mannose}\mspace{14mu}{epitopes}} \right\rbrack}\underset{k_{2}}{\overset{k_{1}}{\overset{\rightarrow}{\leftarrow}}}\left\lbrack {{ConA} - {{mannose}\mspace{14mu}{complex}}} \right\rbrack}{{Ka} = \frac{k_{1}}{k_{2}}}{\frac{\left\lbrack {{Con}\mspace{11mu} A} \right\rbrack}{\Delta\; M} = {\frac{\left\lbrack {{Con}\mspace{11mu} A} \right\rbrack}{\Delta\; M_{m\;{ax}}} + \frac{1}{\Delta\; M_{m\;{ax}}K_{A}}}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

In Equation 1, ΔM_(max) is the maximum binding amount, ΔM is themeasured binding amount at equilibrium, and [Con A] is the originalconcentration of Con A.

The value of K_(A) for the binding between Con A and mannose wasestimated to be (5.6±1.4)×10⁶ M⁻¹ (n=5). This result is in goodagreement with reported literature values [(5.6±1.7)×10⁶ M⁻¹]⁽¹³⁾ andour previous work [(8.7±2.8)×10⁵ M⁻¹ (QCM), (3.9±0.2)×10⁶ M⁻¹ (SPR)].

(2) Detection of E. coli W1485 by mannose-QCM: E. coli W1485 (ATCC®12435™) carries type 1 fimbriae, which is specific for mannose binding.The E. coli cell is about a million times heavier than Con A and typicalantibodies; theoretically the binding between E. coli and mannosereceptor on the QCM surface should lead to a very large response.However, when the mannose modified QCM electrode was exposed to thedifferent concentrations of E. coli W1485 (2.9×10⁷, 9.8×10⁷, 1.6×10⁸,and 2.7×10⁸ cells/ml), only small signals were observed (FIG. 5). Thelinear range is very narrow narrow (i.e. 2.9×10⁷-2.7×10⁸ cells/ml). Thefollowing explanations have been suggested for this unexpectedphenomenon. First, the fimbriae mediated adhesion is relatively weak andflexible. Along with the high mobility of the bacteria, it created largefreedom of movement of the bacteria on the QCM surface. Thisnon-acoustic attachment cannot be easily measured by QCM technique.Secondly, QCM will give a signal only if the above interaction resultsin a net change of mass. The weak and flexible binding of the fimbriaeto the mannose may result in a displacement of one species with another.

Consequently, the surface is only a temporary host to the E. coli andthe net change of mass is very small. Finally, a major issue which mustbe considered in bacterial detection is that of antigenic or phasevariation. Phase variation is the adaptive process by which bacteriaundergo frequent and reversible phenotypic changes as a result ofgenetic alterations in specific loci of their genomes. This process iscrucial for the survival of pathogens in hostile and ever-changing hostenvironments. As a result, type 1 E. coli bacteria might shift from afimbriated phase to a nonfimbriated phase and back spontaneously, whichmight affect the fimbriated E. coli attachment.

(3) Detection of E. coli W1485 by mannose/lectin-QCM: As pointed out byE. V. Olsen, et. al., a piezoelectric mass sensor will not be able toprovide quantitative information for bacterial detection if binding ofbacteria on the sensor surface is neither predominantly rigid norpredominantly flexible. The key is to ensure adequate bacterial bindingby using recognition molecules with high affinity and multiple bindingvalences. Here we take advantage of the fact that bacteria and fungihave chemically distinct surface polysaccharide carbohydrate structuresthat can be recognized by lectins in agglutination studies. We usedlectin Con A as an E. coli adhesion promoter to strongly attach E. colito the mannose so a rigid binding layer would be formed on the QCMsurface (FIG. 1).

In order to prove the above strategy, experimental conditions need to beselected so that we can unquestionably demonstrate that the Con Aadsorbed on the E. coli cell surface facilitates the binding of E. colito the mannose receptor rather than free Con A in the mixture ofbacteria and Con A binding to the mannose receptor. The mannose modifiedQCM surface was immersed in a 100 nM Con A solution for 2 h, then theelectrode was rinsed with PBS buffer to remove the unbounded Con A andthe cell was refilled with fresh PBS buffer containing 1 mM Ca²⁺ andMn²⁺. When the similar concentration of E. coli W1485 was added to thetest chamber in which the mannose modified QCM surface has preadsorbedCon A but no Con A is in the binding solution, only ˜40 Hz frequencyshift was observed which is much smaller than the 250 Hz signal obtainedwhen ConA is also present in the solution phase. Similar results wereobtained by SPR experiments (data not shown). Both study confirmed thatthe agglutination of Con A to the E. coli in solution phase is the keyreason for the signal amplification.

We further studied two experimental conditions. In one, a lowconcentration of Con A was first added to the mannose sensor testchamber. The concentration of Con A added is relatively small so thatthe mannose surface is not saturated based on the Con A/mannose bindingstudy (FIG. 4) and the surface still has a plethora of available mannosebinding sites. When the Con A/mannose binding reached equilibrium, E.coli. W1485 was added. At this time, small amounts of free Con A in thebulk solution will facilitate the binding of E. coli to the mannosereceptor as described in FIG. 1. As shown in FIG. 6, the addition of 100nM Con A to the mannose-QCM generated ˜100 Hz frequency change atequilibrium. The subsequent addition of E. coli provided a large bindingsignal (˜230 Hz). This result shows that the presence of Con A in thebinding solution leads to the signal amplification. Since theinteraction between Con A and E. coli has already been proved by severalstudies, we suggest that Con A in the binding solution aggregates on theE. coli cell walls through binding to their distinct surfacepolysaccharide carbohydrate structures that promotes the formation ofrigid adhesion onto mannose-QCM.

Sensor sensitivity: The mannose modified QCM surface was first exposedto the 100 nM Con A solution for two hours (2 h) to reach the bindingequilibrium, then E. coli W1485 samples ranging from 7.5×10² to 7.5×10⁷cells/ml were injected onto the Con A pretreated mannose-QCM sensorchambers which contain 1 ml PBS with 1 mM Mn²⁺, 1 mM Ca²⁺ and 0.1 mM ConA. Fast and large signal responses were observed. A linear relationshipbetween the frequency shift and logarithm of cell concentration wasfound from 7.5×10² to 7.5×10⁷ cells/ml (FIG. 7), which is four decadeswider than the early mannose alone sensor. The damping resistance in theButterworth-Van Dyke-equivalent circuit was also determinedsimultaneously with the frequency shift for the bacterial binding study.The change of damping resistances for E. coli adhesion experiments inFIG. 6 and FIG. 7 were no more than 1.4%. This suggests that thebacterial attachment was rigid, rather than a viscoelastic behavior.

Table 1 lists the limits of detections (LOD) obtained by the QCMtechnique using antibody or DNA recognition elements as reported in theliterature and via the two methods we used (carbohydrate orcarbohydrate/lectin recognition elements). Compared to the use ofcarbohydrate alone, ˜10⁴ fold improvement in detection limits wereachieved using lectin amplification. Currently, the combinedcarbohydrate/lectin detection methods give similar detection limits toDNA sensors using nanoparticle amplification. Since the detection limitsin our study are experimentally determined, there is still room toreduce it further. Additionally, as evident from the Sauerbrey equationabove, the sensitivity of the QCM sensor increases with the square of f₀and linearly with n; thus by working with crystals of higher f₀ or athigher harmonics, even higher sensitivity and lower detection limits canbe obtained.

TABLE 1 Comparison of QCM biosensors for the detection of E. coli Assayprinciple and Linear ranges References E. coli description LOD(cells/ml) (cells/ml) (1) E. coli K-12 Immunosensor: Anti- ECA 10⁶10⁶-10⁹ antibody crosslinked to PEI precoated surface (6) E. coliO157:H7 Immunosensor: Antibody linked 10³ 10³-10⁸ to MHDA SAM E. coliO157:H7 DMA sensor: Nanoparticle 2.67 × 10² 2.67 × 10²-2.67 × 10⁶amplification Results of the E. coli W1485 Carbohydrate sensor (without 3.0 × 10⁷ 2.9 × 10⁷-2.7 × 10⁸ present invention lectin) E. coli W1485Carbohydrate/lectin sensor  7.5 × 10² 7.5 × 10²-7.5 × 10⁷ ECA:Enterobacterial common antigen; PEI: polyetheneimine; MHDA:16-Mercaptohexadecanoic acid; SAM: self-assembled monolayer.

(b) Sensor specificity: Several control experiments were performed tovalidate the conclusions and to test the sensor specificity. The mannosemodified QCM sensor surface was first exposed to 100 nM Con A solutionfor about two hours (2 h), which allowed Con A to partially occupy themannose surface activity sites leaving free Con A in the bulk solution.When the Con A/mannose binding reaction reached to the equilibrium, thefinal concentration of 7.5×10⁷ cells/ml E. coli W1485 were added andgenerated a large signal response (˜230 Hz) (FIG. 8, Curve A), which wasabout 8 times larger than the direct adhesion of E. coli W1485 onto themannose-QCM alone (˜30 Hz signal, Curve C). To determine whether thesurface bound Con A or the Con A in the binding solution was the majorfactor for the enhanced E. coli W1485 adhesion, the following controlexperiment was performed. First, the mannose modified QCM surface wasimmersed in a 100 nM Con A solution for two hours (2 h), then theelectrode was rinsed with PBS buffer to remove the unbound Con A and thecell was refilled with fresh PBS buffer containing 1 mM Ca²⁺ and Mn²⁺.When the similar concentration of E. coli W1485 was added to the testchamber in which the mannose modified QCM surface has preadsorbed Con Abut no Con A is in the binding solution, only ˜40 Hz frequency shift wasobserved (Curve B). This result confirms that the Con A in the bindingsolution rather than the Con A on the QCM surface played the key role inenhancing E. coli cell adhesion onto the mannose modified QCM surface.

A recombinant antibody 210E scFv-cys modified QCM surface was usedadditionally to exam the specificity of the above system. Recombinantantibody 210E scFv-cys binds specifically to rabbit IgG antigen. Withthe same experimental condition as FIG. 8 Curve A, negligible frequencychange was observed for the addition of E. coli W1485 (Curve D).

Staphylococcus aureus serotype 1, a gram-positive bacterium, was furtherused as a negative control. When Staphylococcus aureus was added to theCon A pretreated mannose-QCM electrode, only a very small signal wasdetected (Curve E).

All the above experiments confirmed that Con A in the binding solutionaggregated onto the E. coli W1485 cell wall thus enhanced thesensitivity and specificity for mannose binding with E. coli W1485 bypromoting the formation of rigid attachment to the mannose modified QCMsurface.

(c) Validating the mannose/lectin QCM sensor for E. coli W1485 detectionby a SPR biosensor: Surface plasmon affinity sensors are based onmonitoring the changes in the effective refractive index of the guidedwaves caused by the interactions of their evanescent field with analytemolecules binding specifically to their reaction partners immobilized onthe sensor surfaces. Even though SPR spectroscopy and QCM are based ondifferent physical phenomena, it is possible to use SPR to validate oursurface chemistry since both techniques are non-label mass sensors andcan theoretically provide information for the binding events occurringat solution metal interfaces. FIG. 9 shows the SPR spectra of thestepwise binding of surface receptor to the target analyte. The surfacewas thoroughly washed after each step and then recorded the spectrum.Little angle shift was observed when E. coli W1485 was directly bound tothe mannose SAM (Curve B). Con A binds to the mannose surface and leadsto 0.41° angle shift (Curve C). However, when E. coli W1485 was added tomannose-Con A surface, negligible angle shift was observed (Curve D)confirming our early rational that the agglutination of Con A to the E.coli in solution phase is the key reason for the signal amplification.Finally, as shown in the FIG. 9 insertion, when the incident angle ofSPR was fixed at 54.32°, and the mixture of E. coli W1485 and Con A wereinjected into the Con A pretreated the mannose SAM chamber in which theconcentration of Con A was fixed to be the same as that in curve C, thereflected light intensity change vs. time shows real time bindingevents. After about 70 min, the chamber was rinsed with PBS and SPRspectrum shows a significant angle shift 0.48° (Curve E). In summary,the amplification of binding between the mannose recognition element onthe surface and E. coli W1485 by lectin Con A was observed by the SPRbiosensor using a similar surface chemistry approach as in the QCMexperiments.

This example illustrates the use of a mannose-SH and ConA system with anSPR biosensor device. A portable SPR biosensor device as described inU.S. patent application Ser. No. 11/581,260 to Xiao and Zeng, filed Oct.10, 2006, incorporated herein by reference in its entirety, was used todetect E. coli K12 with a bound lipid mannose-SH sample layer on thegold metallic film of the device. FIG. 10 illustrates the molecularformula of the mannose-SH as a capture reagent bound to gold (Au) thinmetallic film 60 of the surface plasmon resonance biosensor device 10described above. The mannose-SH molecule has a formula of C₂₅H₅₀O₁₀S anda molecular weight (Mr.) of 542.31. A three-dimensional model of themannose-SH is illustrated in FIG. 11. FIGS. 12A and 12B illustratemannose-SH binding to the gold (Au) surface as the thin metallic film 60of the surface plasmon resonance biosensor device 10, in ethanol. Asillustrated in FIGS. 13A and 13B, the lectin concanavalin A (ConA) bindsto the mannose-SH bound to the gold (Au) surface. E. Coli K12 can bindto the ConA surface if there is ConA in the liquid, as illustrated inFIGS. 14A and 14B.

Conclusion: Routine identification and characterization of toxins andmicro-organisms are commonly performed by biosensors containing eitherantibodies or nucleic acid probes as the detection element. However,neither antibodies nor nucleic acids are necessarily the best or evenmost valuable means of identification. In order to have a realisticchance of detecting and identifying an unknown agent, a vast array ofantibodies would be required. For DNA or RNA biosensors, often theDNA/RNA probe must be known and amplification of the DNA/RNA probe isneeded before immobilization. Taking advantage of the fact that a highpercentage of micro-organisms have both carbohydrates and carbohydratebinding pockets at their surfaces, we demonstrate here that acarbohydrate epitope in combination with a lectin amplification strategyis a real possibility for developing a highly sensitive and specificnon-labeled biosensor for bacteria detection. The mannose QCM biosensorshowed a significant improvement of the sensitivity and specificity forE. coli W1485 detection with a detection limit of a few hundredbacterial cells and a linear range from 7.5×10² to 7.5×10⁷ cells/ml thatis four decades wider than the mannose alone sensor. The change ofdamping resistances for E. coli adhesion experiments were no more than1.4% suggesting that the bacterial attachment was rigid, rather than aviscoelastic behavior. Little non-specific binding was observed forStaphylococcus aureus and other proteins (Fetal Bovine serum, Erythrinacristagalli). Carbohydrate epitopes offer several advantages overantibody/nucleic acid detection of antigens. Carbohydrates possess broadinteraction specificity; carbohydrate recognition could enableidentification of unexpected or even novel agents. Carbohydrates do notdenature or lose activity upon changes of temperature or pH; they arestable and could have long lifetime. Oligosaccharides are smaller thanantibodies; consequently, higher densities of carbohydrate sensingelements could lead to higher sensitivity and less non specificadsorption. A few carefully chosen carbohydrate epitopes in combinationwith additional specificity of lectin-bacteria recognition could providethe desired fingerprinting of a high number of biological agents andhave a propensity to be extremely specific for one particular biologicalagent, possessing minimal cross-reactivity for other agents. Inaddition, combining lectin and carbohydrate SAM recognition allows rigidbinding of bacteria to the QCM sensor surface, which significantlyenhances specificity and sensitivity of detection. The present inventionprovides sensor devices highly suitable for the fast, reversible andstraightforward on-line detection of these analytes at very lowconcentrations in complex samples.

The present invention also provides a two dimensionalcarbohydrate-lectin-array that allows for multitude of discretecarbohydrate protein interactions of bacterial cells to be observedsimultaneously, thus resulting in a high throughput probe of cellsurface carbohydrate and lectin adhesin expression using a non-labeledsensor readout. The array technology is easy to assemble since only thecarbohydrate is immobilized on the transducer surface which avoids theloss of binding activity of lectin due to immobilization. Themeasurement is rapid, sensitive, specific, convenient and label freewhich allows real time monitoring of the dynamic changes of cell surfacecarbohydrate and lectin adhesin expression as well as the detection ofbacteria with extremely high sensitivity and specificity. The label freemicroarray format enables the real time measurement of complicatedcarbohydrate-protein interactions with thousands of unique glycans andlectins while consuming only very small amount of precious reagents. Thehigh density of the immobilized carbohydrate on the array surface notonly enhances the binding sensitivity but also accommodates themultivalent binding. The miniaturized and high throughput nature ofmicroarrays makes them a tool to profile cell surface carbohydrate andlectin adhesin expressions and to deliver high quality, high informationcontent. Since carbohydrate and/or lectin adhesin-cell interactions areubiquitous in nature, this improvement has the potential to impact avariety of important areas.

Thus, functionalized self-assembled monolayers (SAM) of specificcarbohydrates on gold surface using thiol SAM and click chemistry areprovided. Selected monosaccharides and a preferred polysaccharides aremodified to facilitate their coupling to gold transducer surface forcarbohydrate array fabrication.

FIG. 15 shows a schematic diagram of the basic 2-dimensionalcarbohydrate lectin microarray of the present invention describedpreviously. The carbohydrate SAMs developed are used in combination withvarious available lectins for a carbohydrate-lectin microarray.Profiling and detection of eight pathogenic category B bacteria(Diarrheagenic E. coli, Pathogenic Vibrios and Salmonella) and twonon-pathogenic bacteria controls via two compatible orthogonal labelfree transduction mechanisms (i.e. electrochemical Impedancespectroscopy (EIS) and quartz crystal microbalance (QCM)) is described.The sensitivity, selectivity, dynamic range and limit of detection andthe binding pattern of the carbohydrate-lectin arrays are evaluated andvalidated using these 10 bacterial strains.

Using standard fabrication processes, the carbohydrate-lectin arrayplatform is integrated with microfabricated electrodes to incorporateelectrochemical impedance and quartz crystal microbalance readout forhigh throughput, simultaneously profiling of bacterial cell surfacecarbohydrate and lectin adhesin expression. A 5×5 2D carbohydrate-lectinarray is fabricated and tested to obtain the expression pattern of theselected 10 bacteria targets. Subsequently, a larger array thatincorporates oligosaccharides and anti-carbohydrate antibodies can beused.

The biosynthesis and expression of any particular cell membraneconstituent is a function of age and the physiological state of the cellas well as the cellular growth environment. Recently there has beenincreasing evidence that glycosylation plays an important role indiverse biological processes such as cell signaling, celladhesion^(5,6), fertilization^(7,8), proliferation⁹, viral/bacteriainfection, apoptosis and the immune response¹⁰ as well as for manydisease states. However, during the study of cell-cell interactions, theproximity of other cell surface constituents can interfere with theability of receptors to bind the ligand. Thus, when comparing thebinding of a given ligand to cells under varying physiologicalconditions, it is often not possible to determine whether any differenceobserved is due to a change in the numbers of receptor molecules,alternations in the recognition-site, or interference by other surfaceconstituents. As a consequence, unraveling the secrets of glycosylationhas become a key research focus in glycoscience and served as afundamental requirement for both basic and translational research.Efforts toward this end have catalyzed the formation of a Consortium forFunctional Glycomics (http://gycomics.scripps.edu), which aims to“understand the role of carbohydrate-protein interactions at the cellsurface in cell-cell communication”. The identification of a propercarbohydrate scaffold has become the prerequisite to thecharacterization of carbohydrate-protein interactions. Over the past tenyears, a plethora of sugar scaffolds have been explored for studying thecarbohydrate-protein interactions¹¹. At the same time, the field hasseen growing interest in the development of glycoarray technology, i.e.displaying carbohydrates on surface, which aims at mapping thecarbohydrate-protein interaction in a high throughput manner. Methodswere developed to immobilize a variety of oligosaccharides, glycolipids,or glycoproteins to the solid supports to probe the carbohydrate bindingproperties of proteins or cells. Although these glycoarrays providevaluable information about carbohydrate-protein interaction. The maindisadvantage of most of the current glycoarrays is that little islearned about the nature of the interactions and information aboutchanges in glycosylation is difficult to obtain.

A panel of specific monoclonal antibodies, each of which could recognizea unique structure and could be used to readily identify and isolatespecific oligosaccharides within a complex mixture. However, it is wellknown that carbohydrates are poorly immunogenic and production of highaffinity carbohydrate binding antibodies using traditional approaches isexpensive and time consuming. Fortunately, nature has already provided avast number of carbohydrate-binding proteins called lectins. Lectins areproduced by all living things. Many of these lectins are known to beimportant for promoting cellular adhesion and acting as receptors forother glycoconjugates. Most lectins have high affinity interactions withspecific carbohydrate determinants, and thus lectins can be used tocharacterize and isolate glycoconjugates on the basis of specificstructural features, instead of the size or charge of the glycans. Inaddition, lectins can be used in a manner akin to antibodies, inmatrices, or on intact glycoproteins, and to study the biosynthesis ofglycoconjugates.

Many Lectins interact with bacteria by binding to the myriad ofcarbohydrate structures present on the cell surface, e.g. techoic acids,lipopolysaccharides, and peptidoglycans. Lectins are generally specificfor a particular carbohydrate structural motif. Many bacterial speciesmay bind to a single lectin and a single species may bind a variety oflectins with different carbohydrate specificities. This lattercharacteristic has been used to develop lectin arrays to analysis ofbacterial cell surface glycosylation. Dynamic alternations of thecarbohydrate components and distinguish glycopatterns of four E. colibacteria were obtained using a lectin microarray. However, there areseveral limitations for lectin arrays. First, only accessiblecarbohydrate motifs rather than the entire glycome are visualized due tolimited availability of lectins.

Second, there is an inherent poor coating efficiency of lectin proteinsas well as an altered availability of their epitopes due to the surfaceimmobilization-associated features. Third, carbohydrate-lectininteractions are often weaker than antibody-antigen interactions, byperhaps a factor of 10²-10³ from typical antibody equilibriumdissociation constant (K_(D)). Finally, the reliance on traditionaldetection systems such as fluorescence can be a serious drawback sincefalse positive results could be produced due to high backgroundfluorescence. There is an increasing need for new tools that permit fastand systematic investigations of the complex glycobiology inherent incellular systems for well defined scientific studies.

The interaction of lectin to carbohydrate, is not a simple monomericbinding event, but an oligo- or polymeric binding mechanism.¹² Lectinsoften have more than one carbohydrate binding site, and it has beenshown that the occurrence of two simultaneous binding events canincrease the avidity of interaction by more than 100-fold,¹² and even ashigh as 10⁴-fold. The present invention takes advantage of thepolyvalent binding situation of lectin to carbohydrate by integration ofthe two lectin-carbohydrate binding events (i.e. use the same lectin asa bridge to link cell surface glycosylation site to the carbohydrateimmobilized on the sensor surface). The previous examples show that thelectin mediator increases the contact area between cell and carbohydrateon the sensor surface and leads to rigid and strong attachment ofbacterial cells to the carbohydrate immobilized on the QCM transducer.By using lectin Con A as a mediator, over 10⁴ fold increase in bindingsensitivity for binding with an E. coli was observed for a mannosesensor than that of without using Con A mediator, thus significantlyincrease the sensitivity and specificity of bacterial detection.

Bacterial cell surfaces often express both carbohydrate and lectinadhesin structures. Thus, it is highly feasible to apply a carbohydratesensor in a two dimensional manner to patterning both cell surfaceexpressed carbohydrates and lectin adhesins. In the first dimension, thecarbohydrate sensor directly senses the bacterial cell surface lectinadhesins, in the second dimension, the carbohydrate sensor senses thebacterial cell surface carbohydrate structures using lectin mediators.Therefore, the carbohydrate-lectin array involves three binding events:two carbohydrate-lectin recognitions and one lectin adhesin-carbohydraterecognition and allows high information content of bacterial cellsurface carbohydrate and lectin expression profiling with surfacesensitive QCM, SPR, or EIS transducer. The 2 dimensionalcarbohydrate-lectin microarray for profiling bacterial cell surfacecarbohydrates and lectin adhesins is used for detection and specificidentification of bacterial pathogens.

Three approaches have been successfully demonstrated to fabricateversatile carbohydrate SAMs with well-defined structures on the goldsensor surfaces. Shown in FIG. 16, in the first example, α-Galtrisaccharide was tailored with a thiol linker, which facilitates theformation of SAMs on the gold surface (FIG. 16A). The binding betweenα-Gal trisaccharide and anti-Gal antibody was thoroughly studied. In thesecond example, the use of surface clicking reaction was explored,specifically the Cu(I)-catalyzed Huisgen 1, 3-dipolar cycloadditionreaction, for anchoring sugars onto preformed SAM. This fabrication wasbased on pre-formed SAM templates incorporated with alkyne terminalgroups, which could further anchor the azido-sugars to form well-packed,stable and rigid sugar SAMs (FIG. 16B).

In the third example, enzymatic glycosylations and in-vitro enzymaticsynthesis of polysaccharides on a pre-sugar SAM (FIG. 16C) was examined.In order to study the multivalent effects of carbohydrate-lectininteractions, alkanethiol containing glycopolymers (FIG. 16E) andcross-linked surface-grafted glycopolymer (FIG. 16D) were developed.These experiments provided efficient fabrication methods of variouscarbohydrate scaffolds on the gold surface and provide a versatileanalytical tool to investigate the carbohydrate-protein interaction andthe biosynthetic pathways of polysaccharides. It was also demonstratedthat the carbohydrate SAMs in concert with non-labeled transducers (i.e.QCM, SPR and EIS)) offered a promising tool for high-throughputcharacterization of carbohydrate-protein interactions. Such acombination should complement other methods such as ITC and ELISA andprovide insightful knowledge of the corresponding complexglycobiological processes.¹³

Experimental conditions were studied to unquestionably demonstrate thatthe Con A adsorbed on the E. coli cell surface facilitates the bindingof E. coli to the mannose receptor rather than free Con A in the mixtureof bacteria that binds to the mannose receptor. Two conditions werestudied. In one, a low concentration of Con A was first added to themannose sensor test chamber. The concentration of Con A added isrelatively low so that the mannose surface is not saturated based on theCon A/mannose binding study and the surface still has a plethora ofavailable mannose binding sites (FIG. 17 Curves B, C, D); In another,the same concentration of Con A was spiked directly to the bacterialcells (Curve A) and then applied to the carbohydrate sensor surface.FIG. 17 Curves A and B show that there is no difference of theequilibrium amount between adding Con A first on the sensor surface oron the bacterial cell sample. This result is highly significant andallows simple development of carbohydrate-lectin array. Lectin can bespiked into the cell sample, and then added to the array or we can addlectin first to the carbohydrate array then add bacterial sample.

Con A has an isoelectric point of about pH 5 and requires calcium ormanganese ions at each of its four saccharide binding sites. At neutraland alkaline pH, Con A exists as a tetramer of four identical subunitsof approximately 26,000 daltons each. Below pH 5.6, Con A dissociatesinto active dimmers of 52,000 daltons. The study was how ConAmultivalency situation affects its role as a mediator. FIG. 17, curves B(pH 7.2), C(pH1.68) and D(pH5.0) show that at physiological pH 7, whileCon A is a tetramer, gives the highest binding sensitivity. Thus, thehigher multivalency of the lectin, the higher efficiency of conjugationof the lectin to both the cell surface carbohydrate and carbohydratesensing element.

Two lectins (Con A and ECL) were used with a mannose sensor to obtainthe carbohydrate and lectin expression pattern of E. coli 1485 and 086.As shown in FIG. 18, the binding patterns of E. coli 1485 and 086 weredistinguished on the mannose carbohydrate sensor. This verified thefeasibility of the proposed approach.

The multivalency of an antibody was explored to mediate the recognitionof bacterial cell surface proteins. Shown in FIG. 19, a recombinantantibody was immobilized on the gold surface and it binds with rabbitIgG. Using rabbit IgG, it allows multivalent antibody mediatedrecognition of S. aureus surface protein A. Protein A is a cell surfacecomponent of S. aureus. All but 1 of 143 strains of S. aureus werepositive for protein A, whereas all 34 strains of Staphylococcus hyicusand 123 of 127 strains of Staphylococcus intermedius were devoid of thiscell wall component.²⁰ The presence of protein A on the cell wall is anaccepted criterion for the identification of S. aureus. Protein A is awell known Fc receptor which binds to the γ heavy chains, the Fc portionof the immunoglobulin. The sandwich antibody assay allows the specificorientation of Fc capture agents by immobilization through recombinantantibody scFv and consistently increases the analyte-binding capacity ofthe surfaces, with up to 7-fold improvements over surfaces with randomlyoriented IgG capture agents (data not shown). Non-specific binding of E.coli to the scFv sensor is very small (the S. aureus sample waspretreated by 0.1 M Na-citric acid elution buffer (pH 2.8) to remove theIgG bond on the surface). Thus, the proposed carbohydrate microarray canbe easily expanded by incorporating antibodies for lectins orcarbohydrates to profile the whole glycome that can overcome theproblems of limited availability of lectins.

The technology needed for a carbohydrate-lectin array is provided thatallows rapid profiling cell surface carbohydrate and lectin expressionfor bacterial detection. It also provides valuable tool for cellanalysis. For example, antibiotic-induced release of lipopolysaccharides(LPS) also called endotoxin was reported to be an important cause of thedevelopment of septic shock in patients treated with severe infectionscaused by gram-negative bacteria. (ref: van Langevelde, P.; Kwappenberg,K. M. C.; Groeneveld, P. H. P.; Mattie, H.; van Dissel, J. T.“Antibiotic-induced lipopolysaccharide (LPS) release from Salmonellatyphi: Delay between killing by ceftazidime and imipenem and release ofLPS.” Antimicrobial Agents and Chemotherapy 1998, 42, 739-743.: Tamaki,S.; Sato, T.; Matsuhas. M “Role of Lipopolysaccharides in AntibioticResistance and Bacteriophage Adsorption of Escherichia-Coli K-12.”Journal of Bacteriology 1971, 105, 968-). The array can thereforeelucidate the role of antibiotics on the dynamics of bacterialcarbohydrate and lectin expression by an investigation of real timechanges in the carbohydrate and lectin expression patterns. It willprovide a measure of endotoxin release so important to septic shockcomplications in infectious diseases as well as provide essentialinformation towards synergistic use of endotoxins binding andneutralizing agents in conjunction with antibiotics.

There are two key technologies: carbohydrate SAM fabrication andcarbohydrate-lectin microarray development. The results presented aboveshow the glycosurface chemistry, the characterization of thecarbohydrate-protein interaction, the lectin-carbohydrate label freemicroarray platform technology.

TABLE 2 Pathogenic bacterial targets and carbohydrate and lectin arraycomponents. Biosynthetic Bacterial gene cluster isolates O-antigenstructure (Accession #) Binding lectin E. coli O8/9→3)-β-D-Man-(1→2)α-D-Man-(1→2)-α-D-Man-(1→ AB010150 α-Man: Canavaliaensiformis (Con A) E. coli O26→3)-a-L-Rha-(1→4)-a-L-FucNAc-(1→3)-b-D-GlcNAc-(1→ DQ196413 GlcNAc:Datura stramonium (DSA) E. coli O128→6)-b-D-Gal-(1→3)-b-D-GalNAc-(1→4)-a-D-Gal-(1→3)-b-D- AY217096 GalNAc:Helix GalNAc-(1→ pomatia (HPA) E. coli O157→2)-a-D-Per4NAc-(1→3)-a-L-Fuc-(1→4)-b-D-Glc-(1→3)-a-D- AF061251 α-Fuc:Anguilla GalNAc-(1→ anguilla (AAA) GalNAc: Helix pomatia (HPA) S.enterica O35

AF285969 GlcNAc: Datura stramonium (DSA) α-Glc: Canavalia ensiformis(Con A) S. enterica B

X60756 α-Man: Canavalia ensiformis (Con A) S. enterica E1→6)-β-D-Man-(1→4)-α-L-Rha-(1→3)-α-D-Gal-(1→ X60665 α-Man: Canavaliaensiformis (Con A) V. cholerae O139

Y07786 GlcNAc: Datura stramonium (DSA)

Bacterial surface lectin adhesin and carbohydrate structure playimportant role in adhesion and infection. Therefore, they are the idealmodel system to verify the principle. Table 2 lists the carbohydratesand lectins that can be used to assemble the 2 D array as well as 8pathogenic bacterial targets. Two nonpathogenic strains are used (E.coli 086 and 1485) as controls. Several bacterial strains and types areselected that have same lectin binding activity to evaluate and verifythe potential high specificity of the two dimensionalcarbohydrate-lectin array. The difference of lectin adhesin expressionpattern can allow high specific identification of those bacteria withsimilar lectin binding specificity. The glycosylation and lectin adhesinpattern obtained can be used for rapid, point of care detection ofbacteria. Five by five (5×5) arrays using 5 carbohydrates and 4 lectinsto profile 10 bacteria in Table 1 are provided. The assay will make asignificant impact in the glycoscience and in the areas of infectiousdiseases and screening and determination of harmful pathogens forclinical diagnosis and environmental monitoring.

Monolayers of terminated in short oligomers of the ethylene glycol group([OCH₂CH₂]_(n)OH, n=3-6) prevent the adsorption of virtually allproteins under a wide range of conditions. The mechanisms responsiblefor such resistance are not yet completely understood and may varyaccording to the molecular structures of the groups presented at thesurfaces. Therefore, a general “bi-functional” linker as shown is used.This linker consists of polyethylene glycol ([OCH₂CH₂]_(n)OH, n=3-6)portion and saturated alkyl portion (R═(CH₂)₁₁₋₁₄). For thealkanethiolate sugar SAM, the polyethylene glycol port is linked withcarbohydrate ligands, while the alkyl portion is terminated with —SHgroup which will anchor the molecule on the Au surface of QCM sensor.For click chemistry sugar SAM formation, a bifunctional linkerconsisting of a saturated alkyl portion with a thiol group and aclicking site at each end are made (FIG. 20). A disulfite linker withvaried chain length with two tethered propynoyl groups can besynthesized. This common immobilization strategy offers an array of“clicking” functionalized SAMs ready for fabrication with differentclicking sugars. In terms of SAM formation, the disulfites areundistinguished from the free thiol groups, while the former are morestable and easy to handle (FIG. 20C). Another advantage comes from theamide linkage connecting the propynoyl group and the alkyl chain. Anumber of previous reports have shown that the internal amide groupstend to form lateral hydrogen-bonding network that improve the stabilityof the SAM.

In a sugar library, all of the sugar precursors share a common designmotif ethylene-glycol (OEG) with a pendant oligo-tethered either with HSor azido groups as shown in FIG. 20. Detail below is the two methods forsugar precursor synthesis.

Synthesis of Sugar Alkanethiolates

The synthesis of sugar-linker-SH is showed in FIG. 20 The linker (1) isconnected to sugar via a glycosylation reaction promoted by HgBr₂ andHg(CN)₂.

“Click” Chemistry Fabrication Strategy:

Peracetylated Schimdt's sugar donors were coupled with azido-OEG-OH toafford the desired products. Several azido sugars were synthesized usingthis method with the coupling yields in the range of 60-80% (FIG. 20A).Azido sugars are clicked onto the preformed alkynyl groups terminatedmonolayer. The density of the sugar can be controlled by tuning themolar ratio of azido-oligoethylglycol (N₃—OEG-OH, the dilute linker) andazido sugar. To block any unoccupied click sites, the obtained monolayerare further treated with azido-oligoethylglycol linker under theclicking conditions. This click step helps to maintain a highly packedand rigid sugar monolayer.

Carbohydrate-Lectin 2D Array Fabrication and Characterization (Objective2).

The carbohydrate-lectin 2D array fabrication involves development,evaluation and validation of three distinct phases: (1) covalentattachment of carbohydrates to microarray surfaces using above linkingchemistry; (2) lectin tagging of the target bacterial cells; (3)specific binding of target bacterial cells as well as lectin taggedbacterial cells to the immobilized carbohydrates.

Carbohydrate Microarray Fabrication (1 Dimension)

The fabrication of carbohydrate SAM is the pivotal step for theformation of stable robust sensors. Each carbohydrate (i.e. Glucose(Glc), Mannose (Man), Fucose (Fuc), N-acetyl Glucosamine (GlcNAc) andN-acetyl Galactosamine (GalNAc)) on Table 3 is immobilized at optimalconcentration with proper spacing to obtain the highest possible bindingcapacity for the binding protein. Consequently, the two coupling methodsdescribed above, the concentration of the clicking carbohydrate, typesand concentration of diluent spacers and blocking reagents, gold surfaceroughness on the quality of the film are determined to provide theoptimal combination for generation of carbohydrate immobilized layer.The Au quartz crystal is cleaned with 1:1 mixed concentrated acids(HNO₃: H₂SO₄) and dried gently with nitrogen. The dry frequency of themodified Au electrode will be measured at each step of immobilization toestimate the surface coverage.

Gold surface Roughness: The surface roughness can affect the order andrigidity of immobilized sugar SAM. Two Au surfaces (polished andnon-polished) at two frequencies, 10 M Hz and 25 M Hz AT cut quartzcrystal are used. The use of unpolished surfaces allows a measure of howsurface roughness (i.e. crystallographic orientation of Au) affects thequality of immobilization of carbohydrate.

Ligands presenting density and spacing Fine tuning of ligand density isaccomplished by changing the ratio of functionalized OEG-thiol anddiluent OEG-thiol. The strategies are tested to obtain sugar withoptimal spacing by using various length and concentration of dilutetOEG-thiol for the optimum binding with lectin and on effects ofpotential steric hindrance on the binding of lectin.

Surface resistance to nonspecific adsorption: The non-specificadsorption is assayed with three non-carbohydrate binding proteins,fibrinogen, lysozyme and BSA. Fibrinogen, a large (340 kD) blood plasmaprotein that adsorbs strongly to hydrophobic surfaces, is used as amodel for “sticky” serum proteins and lysozyme, a small protein (14 kD,pI=12) that is positively charged under the conditions of the experiment(phosphate buffered saline, PBS, pH 7.2) is often used in model studiesof electrostatic adsorption of proteins to surfaces. BSA has beentraditionally used as blocking protein to occupy any uncovered sensorsurface to eliminate non-specific adsorption. BSA allows evaluation ofthe overall nonspecific adsorption of the sugar monolayers.

Choice of blocking reagents: After the carbohydrate is immobilized withsuitable spacers inserted, if nonspecific adsorption still occurs,blocking agents are added, to further reduce nonspecific adsorption. Theeffects of various blocking reagents on the sensitivity and non-specificbinding of protein with carbohydrate-SAM is determined. Super Block®,ovalbumin, fish gelatin and albumin are used at various concentrationsand times.

Carbohydrate Microarray Characterization

Following each immobilization step as described, the obtained surfaceimmobilized layer is characterized by multi-techniques (e.g. CyclicVoltammetry, Electrochemical Impedance Spectroscopy, ReflectanceAbsorption FT-IR, Attenuated Total Reflectance FT-IR, Ellipsometry, QCMImpedance Analysis, SPR and Atomic Force Microscopy (AFM)). Thecharacterization applies equally to each of the carbohydrate on Table 1to obtain structural, thickness, rigidity, orientation, stability, andsurface coverage information of the immobilized carbohydrate films.

Carbohydrate-Lectin Array Fabrication (Two Dimension)

Using the optimal conditions obtained from above characterization, thecarbohydrates are spotted to the gold sensor chips using an automaticarraying robot or manually. Shown in FIG. 21, for initial 5×5carbohydrate lectin arrays, each horizontal row is spotted withidentical sugar. The number of rows can be easily enlarged to includethe replication experiments. The gold chips are incubated overnight toallow self-assemble process and coupling reaction to complete. Afterwashing, the gold chips are ready for the profiling experiments.

Lectin Tagging of the Target Bacterial Cells for Binding

Bacteria in table 1 are purchased from ATCC and grown in culture. Forexample, the pure culture of Escherichia coli 157, (ATCC® 43895™) isgrown in ATCC® medium at 37° C. for ˜24 h in a shaking incubator. Theviable cell number is determined by conventional agar plate counting.The fully characterized carbohydrate layers are first evaluatedindividually by EIS, QCM and SPR techniques for binding with theirspecific lectin to obtain the calibration curves to determine theoptimal concentration needed to label the targeted bacteria with lectin.For our initial 5×5 carbohydrate lectin arrays, each of the bacteriagrown in the culture are diluted and aliquot in identical five batchsamples. As shown in FIG. 21, four of the five batch bacterial samplesare tagged with a specific lectin (i.e. DSA, HPA, AAA and Con A) at theoptimal lectin concentration for the binding experiments.

Binding and Detection

The carbohydrate microarray are incubated in the appropriate buffer(e.g. PBS). Then 20 μl or less of each of the five bacterial samples areadded to each column of the array respectively (FIG. 21B). The real timefrequency change vs. time is monitored for QCM measurement. Themulti-channel QCM prototype and commercial QCM instruments (Preliminarystudy) are used. For impedance measurement, each bacterial sample isspiked with redox probe 1 mM Fe(CN)₆ ^(3-/4) and the impedance ismeasured using PAR 2263 potentiostat.

Following the incubation for a desired period, a subsequent addition ofthe analyte (i.e. bacterial sample with or without lectin tags) isadded. The real time, label free readout by QCM and EIS allows multipleadditions of samples without washing steps. Therefore, using one chip,multiple quantitative analysis and monitoring bacterial surfacecarbohydrate and lectin expression are preferred in a dynamic real timemanner.

Regeneration of the Chip

For pathogenic bacteria profiling and detection, once the gold sensorchip is exposed to the bacterial sample, it is considered to becontaminated and should be disposed or decontaminated. De-contaminationcan destroy the immobilized receptors and unsuitable for re-use. Ascarbohydrate modified piezoelectric crystals are inexpensive, i.e., itis affordable to use disposable transducers. The reversibility of thebinding reaction is assessed to deliver the feasibility of continuousmonitoring without calibration. The Au-array regeneration step isdeveloped and optimized using a non-pathogenic E Coli Strain W 1485.After each round of experiment, the carbohydrate-bacteria andcarbohydrate-lectin-bacteria complex is dissociated using standardmethods such as a high salt solution or low pH. The condition will notharm the carbohydrate epitopes. This method is not expensive but it cancause significant degradation after several re-uses. Therefore it issafer practice to remove the whole carbohydrate-antigen complex or usefreshly labeled crystals for each assay. Since the SAM template is usedfor immobilization of carbohydrate, the gold surface can be completelystripped through a reductive desorption process (i.e. carbohydratecomplex-S-Au+e+H⁺→carbohydrate complex-SH+Au) and characterized usingelectrochemical technique. The crystals can be cleaned with properreagents and then relabeled with functionalized linkers for the newtarget application. This procedure can be repeated until the goldsubstrate is either too thin or becomes uneven, which will produce poorimmobilization of the linkers.

Large Array Incorporating with Other Sugars and Antibodies

Four oligosaccharides (Sialyl Le^(x), α-2,6 sialoside, GM3 andGal-α,1,3-Gal trisaccharide) are included to observe the pattern (Table3). Glycosyltransferase mediated glycosylation are employed startingfrom the monosaccharides or disaccharides.

TABLE 3 Lectin and carbohydrate specificity Lectin Carbohydrate Maackiaamurensis (MAA) GM3 Sambucus nigra (SNA) α-2,6 sialoside Lotustetragonolobus Sialyl Le^(x) (Lotus A) Griffonia simplicifoliaGalα-1,3-Gal lectin-1-B4 (GSI-B4) trisaccharideSpecifically, using the corresponding glycotransferases and sugardonors, oligosaccharides are synthesized from azido lactose (FIG. 22).Several available anti-LPS carbohydrate antibodies can be added into thecarbohydrate-lectin array.

The carbohydrate-lectin array offers wide flexibility in profiling cellsurface carbohydrate and lectin expression. It overcomes the limitationsof both carbohydrate array and lectin arrays and significantly expandsresearch capacity in glycobiology. They can impact various fieldsincluding bacterial pathogenesis, tumor cell metastasis, andinflammation and provide a valuable tool for bacterial detection forbioterrorism defense, environmental pollutant monitoring, forensicanalysis, biological research, and diagnosis for bacterial infection.

The Anticipated Advantages of the 2D Lectin-Carbohydrate Microarray Are;

1. Selective. Carbohydrate-lectin array is highly selective due to the 2dimensional fingerprints of cell surface carbohydrate and proteinexpression;

2. Sensitive. High density of expressed carbohydrates on bacterial cellsurface and high density of carbohydrate recognition elements on sensorsurface reduce the non-specific interaction and enhance the sensitivity;

3. Accurate. Orthogonal sensing by EIS and QCM reduce false positives.Pattern Recognition by sensor array further increases the accuracy;

4. Real time: QCM is real time and EIS is near real time measurement,allowing results to be obtained very quickly;

5. Non-destructive: The cells can be either harvested for test ormonitored dynamically for other biological studies;

6. Long shelf-life: carbohydrates are more stable and smaller thanantibody/nucleic acid, they rarely denature or lose activity;

7. Simple and low cost. It does not require process treatment to measureand does not require fluorescence labeling. EIS and QCM are low costtransduction mechanisms and ready for miniaturization using microsystemtechnologies permits batch fabrication and low cost manufacturing; and8. Fast. One step detection and miniaturization helps to reduce the timeto reach equilibrium.

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the Claims attached herein.

1. A method of detecting a microorganism in a sample comprising: (a)providing (1) a biosensor device for detecting the microorganism in thesample comprising a solid substrate having a surface covalently bound toa capture agent having a saccharide moiety, (2) the sample, and (3) anunbound lectin capable of binding to the microorganism and to thesaccharide moiety of the capture agent; (b) applying a polyethyleneglycol thiol as a blocking agent to the solid substrate prior toapplying the sample to the solid substrate to reduce nonspecificadsorption to the solid substrate; (c) applying the sample and theunbound lectin to the solid substrate with the capture agent having thesaccharide moiety, for a time to bind the lectin to the saccharidemoiety of the capture agent and the lectin to the microorganism toattach the microorganism to the solid substrate; and (d) detecting themicroorganism attached to the solid substrate with the biosensor device.2. The method of claim 1, wherein the substrate is provided as acomponent in a quartz crystal microbalance (QCM) device.
 3. The methodof claim 1, wherein the substrate is provided as a component in asurface plasmon resonance (SPR) device or an impedance device.
 4. Themethod of claim 1, wherein: (i) the biosensor device comprises an arrayof solid substrates, each solid substrate having a surface covalentlybound to a capture agent having a saccharide moiety, the saccharidemoiety being different for at least some of the solid substrates suchthat the array of solid substrates has a plurality of differentsaccharide moiety types.
 5. The method of claim 1, wherein: (i) thebiosensor device comprises an array of solid substrates, each solidsubstrate having a surface covalently bound to a capture agent having asaccharide moiety.
 6. The method of claim 1, wherein: (i) the biosensordevice comprises an array of solid substrates, each solid substratehaving a surface covalently bound to a capture agent having a saccharidemoiety, the saccharide moiety being different for at least some of thesolid substrates such that the array of solid substrates has a pluralityof different saccharide moiety types; (ii) the unbound lectin comprisesa plurality of different unbound lectin types, each unbound lectin typebeing capable of binding to at least one of the saccharide moiety typesof the array of solid substrates; (iii) applying the sample and theunbound lectin in part (b) comprises applying a portion of the sampleand an unbound lectin type to each of the solid substrates having aselected saccharide moiety type, the unbound lectin type being selectedsuch that there is a plurality of combinations of the saccharide moietytype and the unbound lectin type in the array.